Wnt proteins are secretory proteins discovered as morphogens important for development and morphogenesis in higher organisms. In humans and mice, 19 types of Wnt proteins are known. Wnt proteins are recently recognized to be closely involved in bone formation (Non Patent Literature 1), cancer (Non Patent Literature 2), stem cell maintenance (Non Patent Literature 3), etc., and have drawn attention from not only biological and medical researchers but also pharmaceutical companies. Wnt proteins are strongly hydrophobic due to fatty acid (palmitoleic acid) modification of a specific serine residue (Non Patent Literature 4 and 5), so that they aggregate and denature in an aqueous solution. For this reason, Wnt proteins are widely known to be quite difficult to purify and store. On the other hand, this fatty acid modification is reportedly essential for the biological activity of Wnt proteins and involved in binding of Wnt proteins to Frizzled proteins, which serve as a receptor for Wnt proteins (Non Patent Literature 6). As just described above, Wnt proteins are currently the focus of considerable attention as a research target and tool, but the difficulty in purification and storage has been a major obstacle to studies related to Wnt proteins.
L cells (from mouse fibroblasts), a cell line stably expressing Wnt proteins such as Wnt3a and Wnt5a, have been established and are available from ATCC etc. (ATCC CRL-2647, ATCC CRL-2814, etc.). In the studies to assess Wnt activity, the culture supernatant of such Wnt protein-producing cells is used without purification in most cases. However, the concentrations of Wnt proteins in the culture supernatant cannot be determined, which makes it impossible to achieve quantitative analysis. Moreover, the secretion of Wnt proteins from the cells requires addition of fetal bovine serum to culture medium, and hence fetal bovine serum is inevitably contained in the above-described culture supernatant. For this reason, the culture supernatant of Wnt protein-producing cells cannot be used for assays which are susceptible to serum components (for example, cell differentiation assay etc.). In view of these problems, it would be desirable in various settings to use purified Wnt proteins.
Currently, in an established method for purifying Wnt proteins, the culture supernatant of cells stably expressing Wnt proteins is subjected to more than one step of chromatography (Non Patent Literature 7). Wnt proteins thus purified are commercially available from R&D Systems etc. However, the purification method of Non Patent Literature 7 involves very cumbersome and highly skilled operations as well as requires large-scale protein purification equipment. In addition, this method requires constant addition of a surfactant and a high-concentration salt in the course of purification steps. For this reason, when Wnt proteins purified as above are used, the contamination of Wnt proteins with the surfactant and the salt is a problem. Moreover, Wnt proteins purified by the method of Non Patent Literature 7 are difficult to store in such a manner that Wnt proteins can maintain their activity. In fact, commercially available purified Wnt proteins are expensive, but their activity is too low for the high price. That is, current techniques cannot provide highly purified Wnt proteins of known concentrations in a highly active state in physiological buffer. Therefore, if such Wnt proteins can be provided, many useful studies related to Wnt proteins would be exponentially accelerated.